EPA-670/2-74-018
April 1974
Environmental Protection Technology Series
BACTERIAL ZOOGLOEA
FORMATION
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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EPA-670/2-74-018
April 1974
BACTERIAL ZOOGLOEA FORMATION
by
Richard F. Unz
Samuel R. Farrah
Pennsylvania State University
Department of Civil Engineering
University Park, Pennsylvania 16802
Project No. 17050 DBI
Program Element No. 1BB043
Project Officer
C. W. Chambers
Advanced Waste Treatment Research Laboratory
National Environmental Research Center
Cincinnati, Ohio 45268
Prepared for
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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EPA Review Notice
This report has been reviewed by the EPA, and
approved for publication. Approval does not
signify that the contents necessarily reflect
the views and policies of the Environmental
Protection Agency, nor does mention of trade
names or commercial products constitute
endorsement or recommendation for use.
ii
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FOREWORD
Man and his environment must be protected from the adverse effects
of pesticides, radiation, noise and other forms of pollution, and the
unwise management of solid waste. Efforts to protect the environment
require a focus that recognizes the interplay between the components of
our physical environment — air, water, and land. The National Environ-
mental Research Centers provide this multidisciplinary focus through
programs engaged in
• studies on the effects of environmental
contaminants on man and the biosphere, and
• a search for ways to prevent contamination
and to recycle valuable resources.
In an effort to achieve the foregoing objectives, this project has
attempted to identify the factors involved in bacterial zoogloea formation.
A thorough understanding of the process of floe formation will be an
invaluable aid to future design engineering and wastewater treatment
research.
A. W. Breidenbach, Ph.D.
Director
National Environmental
Research Center, Cincinnati
iii
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ABSTRACT
Activated sludge floes prepared in wet mounts on microscope slides were
observed to sprout typical, finger-like, bacterial zoogloeae by a process
of outgrowth. The rate of extension of finger-like zoogloeae was
typically 501 to 15.0 ^.m per hr and mean cell doubling time was estimated
to be approximately 2 hrs» Finger-like zoogloea formation appeared to
be an aerotactic or chemotactic phenomenon. Photomicrographic and
fluorescent antibody studies showed that the bacterial zoogloeae con-
sisted essentially of the progeny of specific zoogloea-forming bacteria.
Purified exopolymers obtained from axenic cultures of Zoogloea strains
and domestic activated sludge contained two amino sugars, one of which
was identified as glucosamine,, Zoogloea exopolymer consisted of
approximately 17 to 19 per cent amino sugar on a dry weight basis.
Hexoses, uronic acids and ether soluble substances were only about one
per cent of the dry weight of polymer and the polymer was not fibrilar
or affected by reaction with cellulasec Amino sugar production was
found to parallel zoogloea formation by Zoogloea sp.
Calcium ion appeared to augment flocculation of bacterial cells which
were capable of undergoing natural coalescence. Two types of cells,
described as rough and smooth colony-forming, were found in some strains
of Zoogloea0 Rough cells readily flocculated in agitated cultures
whereas smooth cells produced relatively turbid cultures under similar
growth conditions. A predominance of one of the two types could
influence the degree of flocculation by Zoogloea cultures.
This report was submitted in fulfillment of Project Number 17050 DBI by
the Pennsylvania State University under the sponsorship of the Environ-
mental Protection Agency. Work was completed as of October 1973.
IV
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CONTENTS
Abstract iv
List of Figures vi
List of Tables ix
Acknowledgments xi
Sections page
I CONCLUSIONS 1
II RECOMMENDATIONS 2
III INTRODUCTION 3
IV MATERIALS AND METHODS 5
CINEMATOGRAPHY 5
BACTERIA AND BACTERIAL CELL ENUMERATION 5
SEROLOGY 7
ZOOGLOEAL MATRIX (EXOPOLYMER) 8
BACTERIAL FLOCCULATION 10
NATURAL BACTERIAL ZOOGLOEA FORMATION 11
V RESULTS 12
CINEMATOGRAPHY 12
SEROLOGY 21
ZOOGLOEAL MATRIX (EXOPOLYMER) 26
BACTERIAL FLOCCULATION 47
NATURAL BACTERIAL ZOOGLOEA FORMATION 64
VI DISCUSSION 73
VII REFERENCES 77
VIII LIST OF PUBLICATIONS 85
IX GLOSSARY 86
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FIGURES
No. Page
1 Equipment Used for Time-Lapse Cinematography. 6
2 Natural, Finger-Like, Bacterial Zoogloeae. 13
3 Sequential Development of Two Bacterial Zoogloeae
from an Activated Sludge Floe. ^
4 Movement of Bacteria Within a Natural, Finger-
Like, Bacterial Zoogloea. 15
5 Cell Departure from Natural, Finger-Like, Bacterial
Zoogloea. 16
6 Natural, Finger-Like, Bacterial Zoogloea Exhibit-
ing Vast, Cell-Free Regions. 17
7 Portion of Axenic, Finger-Like, Bacterial Zoogloea
Showing Intact and Ghosted Cells. I8
8 Natural, Finger-Like, Bacterial Zoogloea Undergoing
Branching. 19
9 Development of Natural, Finger-Like, Bacterial
Zoogloeae from Activated Sludge Floes. 20
10 Disintegration of a Natural, Bacterial Zoogloea. 22
11 Relationship Between Time and Extension of Two,
Finger-Like, Bacterial Zoogloeae from an Activated
Sludge Floe 23
12 Increase in Number of Bacteria During Extension
of a Natural, Finger-Like, Bacterial Zoogloea
from an Activated Sludge Floe. 25
13 Natural, Finger-Like, Bacterial Zoogloeae Treated
with Zoogloea ramigera 106 Conjugated Antiserum. 28
14 Natural, Finger-Like, Bacterial Zoogloeae and
Filamentous Bacteria Treated with Zoogloea ramigera
106 Conjugated Antiserum. 29
vi
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FIGURES
No. Page
15 Bifurcate, Finger-Like, Bacterial Zoogloea and an
Activated Sludge Floe Treated with Zoogloea
ramigera 106 Conjugated Antiserum. 30
16 Activated Sludge Floes Treated with Zoogloea ramigera 31
106 Conjugated Antiserum.
17 Zoogloeae of Zoogloea MP6. 34
18 Water-Sheared, Cell-Free Exopolymer of Zoogloea MP6. 35
19 Absorption Spectrum of Unhydrolyzed Zoogloea MP6
Exopolymer. 38
20 Comparative Paper Chromatography of Acid Hydrolyzed
Zoogloea MP6 Exopolymer Revealing Spots A, B, and C. 39
21 Paper Chromatography of Acid Hydrolyzed Zoogloea
MP6 Exopolymer and Reference Compounds. 40
22 Column Separation of Amino Sugars Present in Acid
Hydrolyzed Zoogloea MP6 Exopolymer. 43
23 Growth, Flocculation, and Amino Sugar Production
by Zoogloea MP6. 48
24 Development of Zoogloeal Floes by Zoogloea MP6. 49
25 Influence of Cations on Flocculation of Zoogloea
MP6. 50,51
26 Influence of Magnesium Ion on Flocculation of
Zoogloea MP6 and Zoogloea ramigera. 106.
27 Influence of Calcium Ion on Flocculation. of Zoogloea
ramigera 106. 53
28 Colonies of Zoogloea MP6 on Solid Culture Medium. 57
29 Growth and Amino Sugar Production by Zoogloea MP6
at Different Incubation Temperatures. 60
vii
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FIGURES
No. Page
30 Influence of Sodium Thioglycollate on Amino
Sugar Production by Zoogloea MP6. 61
31 Influence of Sodium Ascorbate on Amino Sugar
Production by Zoogloea MP6. 62
32 Influence of Sodium Thiocyanate on Amino Sugar
Production by Zoogloea MP6. 63
33 Chemical and Microbiological Characteristics of
State College Mixed Liquor During Storage. 66
34 Chemical and Microbiological Characteristics of
University Park Mixed Liquor During Storage. 67
35 Scum Layers Harvested from Beakers of Stored,
Fortified Mixed Liquor. 71
viii
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TABLES
1 Extension of Bacterial Zoogloeae from Activated
Sludge Floes 24
2 Specificity of Zoogloea ramigera 106 Antiserum
in Fluorescent Antibody Tests with Axenic Bacteria 27
3 Specificity of Zoogloea ramigera 106 Antiserum in
Fluorescent Antibody Tests with Natural Finger-
Like Zoogloeae Present in Mixed Liquor Scum 32
4 Total and Viable Numbers of Bacteria Released from
Zoogloea MP6 Zoogloeae with Dipotassium Phosphate 32
5 Separation and Recovery of Exopolymer from
Zoogloea MP6 36
6 Separation and Recovery of Exopolymer from Zoogloea
ramigera 115 and _Z. ramigera I-16-M 37
7 Paper Chromatography of Acid Hydrolyzed Exopolymer
Obtained from Zoogloea ramigera 106 and Zoogloea MP6 41
8 Free Amino Sugar Content of Zoogloea MP6 and Z_.
ramigera 106 Exopolymers Hydrolyzed with 6 N HC1
in Boiling Water 41
9 Absence of N-Acetyl Hexosamines in Exopolymer of
Zoogloea MP6 44
10 Chemical Composition of Zoogloea MP6 Exopolymer 45
11 Chemical Composition of Activated Sludge Exopolymer 46
12 Influence of Metal Ions on Flocculation of
Zoogloea MP6, 21, and Z^. ramigera 106 54,55
13 Flocculation and Colonial Morphology (Solid Culture
Medium) of Zoogloea MP6 58
14 Flocculation of Rough and Smooth Cultures of
Zoogloea MP6 59
ix
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No.
15
16
17
18
TABLES
Influence of Carbon to Nitrogen Ratio on Flocculation
and Amino Sugar Production by Zoogloea MP6 65
Physical, Chemical, and Microbiological Characteristics
of Stored Mixed Liquor 68
Influence of Reducing Compounds and Sodium Lactate on
the Formation of Scum at the Surface of Mixed Liquor
Stored in Glass Beakers 70
Influence of Organic Compounds on Scum Production at
the Surface of Mixed Liquor Stored in Glass Beakers 72
x
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ACKNOWLEDGMENTS
The authors are indebted to Mr. G. William Hughes of the Motion Picture
Services Department of The Pennsylvania State University for his
instruction and expertise in cinematography.
The electron photomicrograph (Figure 7) was taken by the late Pauline
Holbert, The Holbert Electron Microscopy Laboratory, Glen Ridge, New
Jersey.
The absence of microfibrils in the exopolymer of Zoogloea strains was
determined from electron photomicrographs taken by Mr. Thomas Rucinsky,
Department of Microbiology, The Pennsylvania State University.
The support of the project by the Office of Research and Monitoring of
the U. S. Environmental Protection Agency and the kind assistance of
the Project Officer, Mr. Cecil W. Chambers, is gratefully acknowledged.
xi
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Section I
CONCLUSIONS
1. Bacterial zoogloea of the finger-like type are formed during growth
and multiplication of zoogloea-forming bacteria which are present in
activated sludge floes.
2. The formation of the finger-like zoogloeae seems influenced by
aerotaxis, chemotaxis or a combination of these phenomena.
3. The gelatinous matrix of a zoogloea does not inhibit movement of
component bacterial cells and it is even possible for cells to escape
from the zoogloeal structures.
4. The gelatinous matrix is an exopolymer containing two amino sugars,
one of which has been identified as glusosamine.
5. Amino sugars are present in fairly constant and similar amounts in
exopolymer of axenic Zoogloea strains. Amino sugars may be used as an
indirect measure of exopolymer and these sugars are from 18-19 per cent
of the dry weight of purified polymer.
6. Two amino sugars were the major reducing substances found in the
purified exopolymer of domestic activated sludge and one of these was
identified as glucosamine.
7. Amino sugars appear in association with the flocculation and
zoogloea formation by Zoogloea strains. Flocculation of cells begins
in the late logarithmic phase of the growth cycle and amino sugars are
produced well into the stationary phase.
8. Natural finger-like zoogloeae consist essentially of the progeny of
a single strain of zoogloea-forming bacterium which most probably belongs
to the genus Zoogloea.
9. Calcium ion may, under appropriate conditions, augment flocculation
of cells of Zoogloea sp. It does not appear that cells which do not
naturally flocculate are positively affected by calcium ion.
10. Smooth and rough colony-forming cells exist in cultures of Zoogloea
strains and it is the rough type cells which strongly flocculate.
11. A depressed oxidation-reduction potential does not appear to
stimulate formation of zoogloeae by axenic Zoogloea strains. In mixed
culture, lowered oxidation-reduction potential creates an environment
conducive to anaerobic decomposition of organic matter and the metabolic
by products (volatile acids) may be utilized by zoogloea-forming
bacteria under microaerophilic and aerobic conditions.
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Section II
RECOMMENDATIONS
The research described in this report was limited essentially to
experimentation with axenic Zoogloea strains and aerobic wastewater
sludges. The study focused on the bacteria which form finger-like
zoogloeae in liquid culture medium. Although it is apparent that these
organisms are present in activated sludge and may be active under
appropriate conditions, it is not known if they contribute significantly
to the formation of activated sludge. A means to quantify viable
Zoogloea spp. in sludges is desparately needed in order to determine
their numbers in floes originating under different cultural conditions.
A screening of numerous bacteria, freshly isolated from activated
sludge, should be undertaken to identify the zoogloeal organisms and
perform chemical analyses on the exopolymers. Since it may be expected
that exopolymers of sludges are the products of resident microorganisms,
it may be enlightening to "finger print," by chemical characterization,
the polymers of various sludges. Such an undertaking may lead to the
development of a new procedure for determining the quality of sludges
in relation to wastewater treatment performance and be of assistance in
maintaining control of aerobic biological treatment systems. Samples
of mixed liquor suspended solids should be obtained from several
different biological wastewater treatment plants and examined for the
presence of finger-like zoogloeae in floes. In this way, it may be
possible to establish a correlation between finger-like zoogloeae in
activated sludge and some feature(s) or condition(s) of treatment plant
operations. The finger-like zoogloeae may prove to be a sensitive
indicator of changing environmental conditions in the activated sludge
process.
The important mechanisms of flocculation and deflocculation in activated
sludge have not been resolved and further study in this area is highly
desirable. The discovery of rough and smooth colony-forming cells in
cultures of Zoogloea strains has far reaching implications because if
these bacteria are important structural determinants of activated sludge
new approaches to the study of wastewater bioflocculation will be in
order. It would be of interest to learn if other bacteria isolated
from activated sludge give rise to rough and smooth cells.
Finally, refined growth experiments need to be performed with Zoogloea
strains. . Chemostat studies employing a suitable substrate would be
valuable for determining minimum nutrient levels required to sustain
maximum growth of the Zoogloea sp= Continuous culture should be used
to find the conditions under which Zoogloea sp. will grow and flocculate
in chemically defined culture media and wastewaters.
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Section III
INTRODUCTION
Bacterial zoogloeae consist of bacterial cells embedded in a confining
gelatinous matrix. The importance of the zoogloeal masses to the
structure of activated sludge has been emphasized in the past (10, 30).
However, other mechanisms have been advanced to explain bioflocculation
leading to the formation of activated sludge (13, 15, 43, 44, 53, 57).
The validity of certain flocculation theories has been challenged (16,
57, 63). It should be noted that zoogloea formation and bacterial
flocculation are not always similar (23, 64).
The floe-forming pseudomonad, Zoogloea ramigera, has been intimated as
an important functional bacterium in the activated sludge and trickling
filter wastewater treatment process (10, 11, 12, 29, 33). Z_. ramigera
was originally described and named in 1867 by Itzigsohn (31). The species
designated was made on the basis of the finger-like or tree-like
zoogloeae which distinguished the organism in mixed cultures from
another less obvious zoogloea-forming bacterium, "L_, termo. Concern
about the taxonomic validity of Z_. ramigera and its significance in
aerobic biological wastewater treatment has inspired several laboratory
studies on microorganisms identified as Z_. ramigera. The information
obtained from these investigations has been largely inconclusive in
clarifying the position of Z_, ramigera with respect to the important
aforementioned issues. Conflicting reports on the characteristics of
assumed pure cultures of Z_. ramigera increased confusion about the
organism. It remained to obtain bacterial cells from wastewater
zoogloeae in a manner which would permit definite statements about the
origin of isolates. Such experiments were performed by Unz and Dondero
(65) who showed that the majority of the bacterial cells present in
finger-like bacterial zoogloeae were able to form in axenic cultures
zoogloeae which were similar in appearance to the natural wastewater
forms. The axenic cultures of zoogloea-forming bacteria were character-
ized and found to be dentrifying, ureolytic pseudomonads which could
hydrolyze gelatin and grow readily on short chain fatty acids but were
inactive on carbohydrates (65, 66). These bacteria were identified as
strains of Zoogloea which, in many ways, were dissimilar to the non-
zoogloeal, floe-forming "L* ramigera I-16-M of Crabtree and McCoy (14)
and the Z. ramigera 115 of Freidman and Dugan (23). Unz (64) suggested
adoption of J2. ramigera 106 (ATCC 19544) as the neotype of Z_. ramigera
to replace Z_. ramigera I-16-M (ATCC 19623).
Unz and Dondero (67) have isolated nonzoogloea-forming bacteria from
finger-like and amorphous wastewater zoogloeae. They found several of
these bacteria to be active in degradation of carbohydrates and they
proposed that some wastewater zoogloeae may consist of certain bacteria
responsible for the formation of zoogloeal matrix as well as non-
zoogloeal bacteria which become entrapped in the zoogloeal matter during
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expansion of the floe. No definite answers are available to support
these hypotheses and further research on the mechanisms of bacterial
zoogloea formation is needed. In an attempt to learn more about the
nature of bacterial zoogloea formation, a research program was under-
taken to study the development of bacterial zoogloeae in mixed cultures
and elucidate the factors stimulatory to growth and zoogloea production
by Zoogloea sp.
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Section IV
MATERIALS AND METHODS
CINEMATOGRAPHY
Preparation of specimens for photographing bacterial zoogloea formation.
A suspension of activated sludge floes was mounted on a glass microscope
slide and covered with a 22 x 44 mm coverslip which was sealed with
vaseline. Approximately one-third to one-half of the total volume
beneath the coverslip was occupied by intentionally entrapped air. A
satisfactory density of sludge particles for microscopic observation was
achieved by trial and error. It was found that very high and very low
concentrations of sludge particles were unsuitable for obtaining good
bacterial zoogloea formation. Slides were observed at ambient temper-
ature (28 C + 1 C) .
Cinematographic techniques .
Wet mounts of activated sludge floes were examined using a Zeiss
Universal microscope equipped with phase optics (Carl Zeiss, Inc.,
New York; Fig. 1, A). Time lapse cinematography was performed using an
Arriflex model S 16 mm motion picture camera with an Arriflex DOM single
frame drive motor (Arriflex Corp., West Germany; Fig. 1, B). The camera
was attached to a Wild microscope stand (Wild Microscope Co., Heerbrugg,
Switzerland; Fig. 1, C). Continuous illumination of slides was avoided
by the use of an Ilex electronic shutter no. 4 mounted over the field
diaphragm (Ilex Optical Co., Rochester, N. Y.; Fig. 1, D). Exposures
were taken at the rate of four to six frames per minute. Timing was
controlled by a time lapse intervalometer (Camera Equipment Co., Inc.,
New York; Fig. 1, E). Coordination of the camera and the electronic
shutter was implemented by a camera-shutter function timer fabricated
at the Pennsylvania State University. Constant voltage for the light
source was maintained with a Solatron model 2KVA voltage regulator (Sola
Electric Co., Elk Grove Village, 111.)
Still photographs were taken with a 35 mm Zeiss Ikon attachment camera.
BACTERIA AND BACTERIAL CELL ENUMERATION
Axenic cultures of the following bacteria were employed in experiments:
(a) Zoogloea strains 9, 21 (ATCC 19122), 106 (ATCC 19544), 201 (ATCC
19325), 216 (ATCC 19123), 235 (ATCC 19324), 239 (ATCC 19173), I-16-M
(ATCC 19623), ^_. ramigera 115, which was kindly supplied by P. R. Dugan,
The Ohio State University, Columbus, Ohio, freshly isolated Zoogloea
strains (68) and; (b) various bacteria from the stock culture collection
of the Department of Microbiology, The Pennsylvania State University.
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Fig. 1. Equipment Used for Time-Lapse Cinematography. A, Zeiss
Universal microscope; B, Arriflex DOM 16 mm camera; C,
Wild microscope stand; D, Flex electronic shutter; E,
time-lapse intervalometer; F, camera shutter function
timer.
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Stock cultures of Zoogloea strains were maintained in liquid Casitone-
yeast autolysate (CY) medium containing per liter of distilled water:
Casitone (Difco), 5.0 g. and yeast autolysate (Charles Pfizer and Co.,
Inc., New York), 1.0 g.
Direct cell counts were made at 500 X using a Petroff-Hausser counting
chamber with the aid of a Zeiss Universal microscope.
Viable cell counts were determined by spread plating appropriate dilu-
tions of culture fluids on CY medium. Plates were incubated at 23 C
for 72 hr.
Dry weight of twice washed biomass was estimated by drying samples at
105 C and cooling to constant weight.
Cell .nitrogen was determined by the semimicro-Kjeldahl method of
McKenzie and Wallace (42).
Optical density measurements were performed at 500 nm using a Bausch &
Lomb Spectronic 70 spectrophotometer (Bausch & Lomb Co., Rochester,
N. Y.).
SEROLOGY
Preparation of inoculum and serum production.
£. ramigera 106 was cultured in a medium containing per liter of dis-
tilled water: (NH,)2SO,, 0.264 g; K^PO^, 0.087 g; MgSO^, 0.120 g;
GaSO,, 0.136 g; sodium lactate, 1.0 g; Casitone, 0.10 g; yeast autolysate,
0.02 g. Incubation was at 20 C on a gyrotary shaking machine for 48 hr.
Four liters of culture fluid were centrifuged at 5000 X g for 10 min
to recover cell mass. Cells were washed twice in distilled water,
suspended in a 50 ml volume of distilled water and the suspension was
adjusted to pH 10.0 using 1 N NaOH. The suspension was boiled for
3 min, cooled, pH readjusted to 10.0 and boiled for another 3 min.
Finally, cells were centrifuged at 27,000 X g for 10 min and washed
twice in distilled water. Microscopic examination of treated cells in
wet mounts containing India ink revealed clean preparations of cells
devoid of exocellular gelatinous substances. A quantity of the clean
cells equal to 1*0 mg Kjeldahl nitrogen per ml was mixed with an equal
volume of mineral oil. The mixture served as the inoculum for the pro-
duction of antiserum. Domestic rabbits received 1 ml subcutaneous
injections in each flank. After 1 month, rabbits were given intravenous
injections consisting of 1 ml of the cell preparation equal to 0.2 mg
Kjeldahl nitrogen per ml. One week following intravenous injections,
antiserum was obtained by bleeding the animals from the heart.
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Preparation and use of flucrescein labeled antibody.
Serum globulins were fractionated from the rabbit antiserum and
buffered according to the procedure of Romano and Geason (55) -
Buffered globulins were mixed with 0.015 mg of fluorescein isothio-
cyanate per mg of protein as suggested by Olson (51). The mixture
was allowed to react overnight at 5 C under continuous mechanical
stirring followed by dialysis against buffered saline consisting of
0.15 M NaCl and 0.1 M BUHPO, ; final pH 7.2. Dialysis was continued
until no fluorescein was observed in the dialysate. The labeled
globulin was sterilized by passage through a 0.45 ym membrane filter
and frozen. Labeled globulin was used in studies of cross reactions
with various known strains of Zoogloea and other axenic cultures of
bacteria as well as unknown bacteria isolated from activated sludge.
The bacteria were isolated on CY agar medium and activated sludge agar
prepared according to the method of Prakasam and Dondero (54).
Bacteria were spread on microscope slides, air dried, fixed in ethanol,
and permitted to react with antiserum for 15 min at 28 C. Following
incubation, slides were washed with buffered saline, covered with
mounting fluid (mixture of equal volumes of glycerol and buffered
saline) and coverslips, and sealed with nail polish. Slides were
examined by dark field microscopy using a Zeiss Universal microscope
equipped with an Osram HBO 200 watt ultraviolet light source and
exciter filter UG 5 and barrier filter 47/65. A cross reaction with
Z. ramigera 115, a bacterium of questionable identity in our opinion,
necessitated further refinement of the Z_. ramigera 106 antiserum before
the antiserum could be used in the analysis of unknown bacterial cells.
Cells of _Z. ramigera 115 were cultivated in CY medium, harvested and
washed as previously described, and suspended in saline to give a final
concentration of 1 mg of cells per ml. To insure the specificity of
the antiserum, equal volumes of Z_. ramigera 115 in saline and labeled
antiserum were incubated at 37 C for one hour. Cells with sorbed
antibody were removed from the antiserum by centrifugation at 27,000 X g
for 10 min. followed by filtration of the purified antiserum through a
0.45 ym membrane filter.
Highly specific Z_. ramigera 106 antiserum was used in diagnostic tests
on the microbial film which formed at the surface of settled activated
sludge stored in beakers at 28 C for 48 hr.
ZOOGLOEAL MATRIX (EXOPOLYMER)
Harvesting, purification, and hydrolysis^
Mass production of the zoogloeal matrix required for chemical analysis
of the polymer was accomplished by batch culturing zoogloea-forming
bacteria in 1 liter quantities of liquid medium on a reciprocating
shaking machine at 20 C. The bacteria were cultivated on several kinds
of media including CY, trypticase soy (BBL) and a basal medium (BM) which
contained per liter of distilled water: (NH.KSO, , 0.264 g; K^HPO, ,
0.087 g; MgSO,, 0.006 g; and sodium lactate, 17000 g to which was added
CaS04, 0.136 g.
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Zoogloeal floes were harvested from 48-hr old cultures by centrifugation
and washed twice in distilled water and suspended in 10 ml of distilled
water. Exopolymers were dissolved either by adjusting the washed
zoogloeae to pH 10 with 1.0 N NaOH or 0.02 M K^HPO, and heating for 10
min in a boiling water bath or by employing a Waring blender to homog-
enize for 1 min 50 ml quantities of the zoogloeae suspended in 0.02
M K2HP04 or distilled water. Cell residue was removed from the dissolved
polymer by centrifugation at 27,000 X g for 10 min. The clear supernatant
was dialyzed against distilled water for 24 hr at 5 C and concentrated
with polyethylene glycol when necessary. The polymeric substance was
precipitated by adding 0.2 g of cetyltrimethylammonium bromide (CTAB)
to 25 ml of solution and dried on standing at 5 C overnight. The
residue was dissolved in 0.5 M NaCl and centrifuged at 27,000 X g for
10 min. The clear supernatant was dialyzed against several volumes of
distilled water over a 24 hr period at 5 C.
Purified, dissolved zoogloeal matrix (approximately 0.5 mg per ml) and
concentrated HC1 were mixed to produce a 6 N HC1 solution which was
dispersed to screw capped test tubes. The matrix was hydrolyzed in a
boiling water bath for 0.25, 0.75, 2.0, 6.0, and 12.0 hr. The HC1 was
removed following hydrolysis by drying samples under a stream of air
at 28 C.
Natural activated sludge was blended in distilled water to obtain
exopolymer which was concentrated and hydrolyzed by the procedures
described above.
Chromatography
Ion exchange chromatography. Zoogloeal matrix hydrolysates were
fractionated in 0.9 by 33.0 cm columns containing Dowex 50 (X8; H
form) resin (J. T. Baker Chemical Co., Phillipsburg, N.J.) according
to the method of Gardell (27). One milliliter fractions were
collected at the rate of 1 to 2 ml per hr using a Warner-ChiIcott model
1205 fraction collector (Warner-Chilcott Laboratories, Richmond, Calif.)
Paper chromatography. One dimensional descending paper chromatography
of hydrolysates was performed using Whatman no. 1 filter paper and one
of the following solvent systems: butanol-acetic acid-distilled water
(12:3:5); butanol-pyridine-distilled water (3:2:1.5); and isopropanol-
distilled water (4:1). Chromatograms were developed for detection of
carbohydrates using a silver nitrate reagent (61) and 3.0 per cent
p-anisidine'HCl in butanol (47). Amino sugars were detected by spray-
ing chromatograms with 0.3 per cent ninhydrin in acetone.
Chemical Analyses. Hexosamine was determined by the modified Elson-
Morgan method as described by Kabat and Mayer (34) employing
D-glucosamine-HCl as the standard. Total reducing sugar was analyzed
by the procedure of Nelson (48) with either D-glucose or D-glucosamine-HCl
as the standard. Uronic acids were determined by the method of Dische (19).
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Hexoses were measured using the phenol sulfuric acid method of Dubois
et al. (20). N-acetyl form of hexosamines was determined by the method
o? Amlnoff, Morgan and Watkins as given by Rabat and Mayer (34).
Volatile acids were determined using column partition chromatography
(3).
Oxidation-reduction potential measurements were made using an Orion
model 801 digital pH/mv meter (Orion Research Inc., Cambridge, Mass.)
equipped with a platinum thimble electrode (Beckman Instrument Co.,
Fullerton, Calif.). ZoBell solution (28) was used to standardize the
system for redox measurements.
Electron Microscopy. Specimens were examined with a Phillips model 300
electron microscope at 60-80 kv. Shadow and replica preparations were
made as described below using a Balzers model BA 360M freeze etch
device (Balzers, Principality of Leichtenstein).
Negative stain. One drop of sample was applied to a carbon-
coated, 200 mesh grid and stained with either one or two per
cent phosphotungstate at pH 7.0-7.2.
Shadowing. One drop of sample was placed on a carbon coated
grid and either drained of excess water before air drying or
permitted to dry completely without prior removal of fluid.
Platinum carbon shadowing was done at an angle between 15 to
30 degrees.
Replica. Air dried samples on clean microscope glass slides
were shadowed at between 15 to 70 degree angles, coated with
carbon at 90 degree angle and floated from the slides.
Cellular material was removed from replicas by treating with
70 percent H^SO, and 0.5 per cent NaOCl for 30 to 60 min
each with intermediate and final rinsing three times with
distilled water.
BACTERIAL FLOCCULATION
Inorganic ions
Various salts were evaluated for their effect on the flocculation of
bacteria. Sulfates and chlorides of Mg, Ca, Zn, Mn, Fe and Na as well
as NaBr, and NaF were tested in concentrations of 0.001, 0.01, 0.1,
and 1.0 mmol per liter. The culture medium used was the BM medium
earlier described.
Carbon to nitrogen ratio
The influence of carbon to nitrogen ratios on bacterial flocculation
was evaluated using BM medium supplemented with 1 mmol per liter of
10
-------
CaSO,. The sole carbon source, sodium lactate, and sole nitrogen
source, ammonium sulfate, were varied in concentration to give a range
of carbon to nitrogen ratios in the culture medium.
NATURAL BACTERIAL ZOOGLOEA FORMATION
Studies on enrichment cultures of branched and finger-like wastewater
zoogloeae were conducted using beakers of activated sludge as previ-
ously described in this report and elsewhere (4, 63). Changes in the
chemical properties of standing volumes of settled activated sludge
were noted by measuring redox potential and the appearance of volatile
acids. Mixed liquors used in these experiments were collected from
wastewater treatment plants located at State College, Pa., and
University Park, Pa. The effect of various reducing agents on the
formation of bacterial zoogloeae we're analyzed by first adding
approximately 40 ml of warm molten agar containing a specific reducing
compound to a 400-ml beaker and, following solidification of the agar
at the bottom of the beaker, admitting 160 ml of mixed liquor over the
agar layer. Specific reducing compounds employed in these experiments
are shown in Table 17. The extent of bacterial zoogloea formation
was determined by harvesting the zoogloea film which, if present,
formed at the surface of beaker fluid in 48 to 72 hr. The film was
carefully skimmed from the liquid surface with a glass microscope slide,
transferred to a centrifuge tube, and concentrated by centrifugation
at 10,000 X g for 10 min. Concentrated films were washed twice and
suspended in distilled water. A measured volume of the suspension was
retained for dry weight determination. The remaining mass was boiled
in 0.02 M K2HPO, at pH 10.0 as earlier described. The extracted gel
was hydrolyzed for 45 min in 6 N HC1, air dried, and analyzed for amino
sugar content.
11
-------
Section V
RESULTS
CINEMATOGRAPHY
Finger-like and branched bacterial zoogloeae were found by microscopic
observation to be present in slimes collected from trickling filters and
in the films and scums which developed at the surface of mixed liquors
stored in beakers (Fig. 2). The bacterial zoogloeae developed by out-
growth from cells present in activated sludge floes which were contained
in wet mounts on microscope slides. The growth of the zoogloeae were
recorded by time-lapse cinematography and the film is available from the
authors of this report. Salient excerpts of the motion picture are pre-
sented herein. Sequential development of finger-like zoogloeae from an
activated sludge floe may be seen in Fig. 3. Apparently, individual
bacterial cells are not rigidly fixed in the gelatinous matrix of the
zoogloea and these may travel within the matrix creating "cell free"
regions (Fig. 4). Bacterial cell 1 and bacterial cell 2 traveled within
the zoogloea in opposite directions. Since cell 2 moved opposite to the
direction of zoogloea extension and the rate of movement for both cell 1
(23 ym per hr) and cell 2 (29 ym perhr) was greater than the extension
rate of the zoogloea (17 ym per hr), it does not appear that changes in
positions of cells was due to physical displacement by stretching or
intercalary expansion of the zoogloea. Bacteria were observed to move
freely within zoogloeae on several other occasions and it was possible
for cells to depart from a zoogloea (Fig. 5).
Apart from the effect of cell movement, cell vacancies in a zoogloea may
result from death and lysis of bacteria. The finger-like zoogloea shown
in Fig. 6 appears to contain bacteria only in the anterior region. The
striking contrast between intact and lysed bacteria in the zoogloea is
evident in Fig. 7.
Branched zoogloeae were frequently observed in natural slimes and in
the scums which developed at the surface of mixed liquors stored in the
laboratory. However, only rarely were branched zoogloeae seen to form
in wet mounts of activated sludge floes and the structures were never
photographed in time lapse studies. However, formation of a bifurcate
bacterial zoogloea (Z2) in a wet mount of activated sludge floes was
photographed with a still picture camera (Fig. 8). It can be seen that
the lateral branch of zoogloea Z2 and zoogloea Zl developed in a
direction contrary to that of the main stem of zoogloea Z2 and zoogloea Z3.
Possibly, zoogloea Zl and the branch of zoogloea Z2 were influenced in
their development by changes in the microenvironment of the microscope
slide culture. Inspection of a composite view of several photographed
microscope fields of a wet mount of activated sludge floes (Fig. 9)
revealed the tendency for bacterial zoogloeae to develop in a direction
12
-------
Fig. 2. Natural, Finger-Like, Bacterial Zoogloeae. Specimen obtained
from scum layer which developed on the surface of mixed
liquor stored in a beaker; 48 hr, 28 C. Zoogloeae treated
with India ink to accentuate matrix boundary. Phase contrast;
wet mount. Bar equals 10 ym.
13
-------
Fig. 3. Sequential Development of Two Bacterial Zoogloeae from an
Activated Sludge Floe. Note appearance of one zoogloea
seemingly from within floe (F, arrow). Time elapsed (min)
A, 0; B, 64; C, 93; D, 138. Phase contrast; wet mount.
Bar equals 10 ym.
14
-------
Fig. 4. Movement of Bacteria Within a Natural, Finger-Like, Bacterial
Zoogloea. Time elapsed (min): A, 0; B, 5.25; C, 8.75; D, 21.
Arrows indicate position of motile cells 1 and 2 within the
zoogloea. Phase contrast; wet mount. Bar equals 10 urn.
15
-------
Fig. 5. Cell Departure from Natural, Finger-Like, Bacterial Zoogloea. Time elapsed (min):
A, 0; B, 6.45; C, 7.45; D, 8.0. Arrows indicate position of cell 2 with respect to cell 1
until its escape from the zoogloea. Phase contrast; wet mount. Bar equals 5 ym.
-------
Fig. 6. Natural, Finger-Like, Bacterial Zoogloea Exhibiting Vast,
Cell-Free Regions. Cells (C) and background debris (D).
Specimen obtained from scum layer which developed on the
surface of mixed liquor stored in a beaker; 72 hr, 28 C.
Zoogloea treated with India ink to accentuate matrix
boundary. Phase contrast; wet mount. Bar equals 10 ym.
17
-------
Fig. 7. Portion of Axenic, Finger-Like, Bacterial Zoogloea Showing
Intact and Ghosted Cells. Electron photomicrograph. Bar
equals 5 urn.
18
-------
Fig. 8. Natural, Finger-Like, Bacterial Zoogloea Undergoing Branching.
Note lateral branch of zoogloea (Zl) developed parallel to
another elongating zoogloea (Z2) whereas zoogloea (Z3) did not
increase in length during the entire 90 minute viewing period.
Time elapsed (min): A, 0; B, 60; C, 75; D, 90. Phase contrast;
wet mount. Bar equals 20 um.
19
-------
I O O u m
Fig. 9. Development of Natural, Finger-Like, Bacterial Zoogloeae from
Activated Sludge Floes. Note zoogloeae (Z) extend from floes
(F) towards interface which exists between entrapped air and
water (arrow, far right). Phase contrast; wet mount. Field
of view: 10° ym.
20
-------
towards an interface formed between water and a small volume of air
entrapped beneath the coverslip of the wet mount. The picture is
suggestive of an aerotactic or chemotactic response by the bacterial
zoogloeae. For unapparent reasons, bacterial zoogloeae may cease to
continue extended growth in the early stages of development ultimately
resulting in disintegration of the colony and dispersion of the bacteria
(Fig. 10). The act may reflect a mechanism whereby the bacteria enter
a swarm stage of behavior, ultimately resulting in the establishment of
new zoogloea colonies through multiplication of the "swarmers". The
factors stimulatory to the onset of a swarm stage were not determined
but may entail conditions, e.g., nutrient deficiency, which are
unfavorable for continued multiplication of the bacteria in the zoogloea.
Finger-like zoogloeae protruded from activated sludge floes at a linear
rate of extension (Fig. 11). Zoogloeae which grew outward from a single
floe extended at similar rates whereas the development of zoogloeae from
heterogeneous floes often occurred at different extensions rates possibly
indicating a sensitive effect of the microenvironment on the bacteria
in the floes or that the zoogloea-forming bacteria in the floes differ
from each other in some respects. The extensions rates of 30 finger-like
zoogloeae were calculated from data obtained during microculture studies
(Table 1). Seventy percent of those zoogloeae measured extended from
activated sludge floes at rates ranging from 5.1 to 15.0 ym per hr.
It was possible, by single frame analysis of the motion picture, to
enumerate bacteria contained in certain developing, finger-like zoogloeae.
Increases in cell numbers were found to be linear with time (Fig. 12)
and the mean doubling time estimated for dividing cells seen in four
zoogloeae was 2.0 hr. This generation time is remarkably similar to
that obtained by axenic Zoogloea strains growing on CY medium in batch
culture at 28 C (unpublished data).
SEROLOGY
Five of eight Zoogloea strains originally isolated by micromanipulation
from natural finger-like zoogloeae fluoresced when stained with
fluorescein labeled antiserum recovered from rabbits inoculated with
cells of Z_. ramigera 106. Only three of 34 Zoogloea strains isolated
by streak plating wastewater samples of sodium m-toluate medium expressed
a similar reaction. A slight antigen-antibody reaction took place
between the labeled serum and Z_. ramigera 115, however, further refine-
ment of the antiserum successfully eliminated the cross reaction.
Purification of the antiserum did not alter the intense fluorescence
which occurred in reaction with slide smears of directly isolated
Zoogloea strains. No cross reaction took place between the antiserum
and Z. ramigera I-16-M, Streptococcus faecalis, Staphylocoecus aureus t
Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, P_.
f lucres cens, and P_. put Ida. In addition, serological tests were con-
ducted on 71 axenic cultures of unidentified bacteria obtained from
21
-------
Fig. 10. Disintegration of a Natural, Bacterial Zoogloea. Time
elapsed (min): A, 0; B, 67; C, 166; D, 210. Phase contrast;
wet mount. Bar equals 10 ym.
22
-------
15 _
N5
UJ
0,5
1.5
Hours
Fig. 11. Relationship Between Time and Extension of Two, Finger-Like Bacterial Zoogloeae
from an Activated Sludge Floe.
-------
Table 1. Extension of Bacterial Zoogloeae from Activated
Sludge Flocsa
Extension rate,
Um/hr
(Class intervals)
Number of
zoo.gloeae
observed in each
class interval
Percentage of
zoogloeae in
each class
interval
0 -
5.1 -
10.1 -
15.1 -
20.1 -
25.1 -
30.1 -
35.1 -
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
2
13
8
5
0
1
0
1
7
43
27
17
0
3
0
3
Total number of zoogloeae observed: 30.
24
-------
K3
t_n
2.0
Fig. 12. Increase in Number of Bacteria During Extension of a Natural
Finger-Like, Bacterial Zoogloea from an Activated Sludge Floe,
-------
mixed liquor and scums formed at the surface of mixed liquors stored in
glass beakers. These bacteria were isolated on CGY and activated sludge
extract media and included 59 gram negative rods, 9 gram positive rods,
and 3 gram positive cocci. None of the 71 unidentified cultures
exhibited cross reaction with £. ramigera 106 labeled antiserum (Table 2).
The purified Z_. ramigera 106 labeled antiserum was seen to react with
certain naturTl, bacterial zoogloeae obtained from the scums of stored
mixed liquors. The affinity of the serum for the zoogloeae was apparent
when treated smears were examined by tungsten (Fig. 13A) and ultra-
violet light (Fig. 13B). Only bacterial cells which reacted with the
antiserum were visible by ultraviolet fluorescence whereas many types
of cells were seen in and around the zoogloeae under tungsten light.
In another series of exposures with labeled serum-treated specimens,
filamentous bacteria and the bacteria of finger-like zoogloeae were
visible by tungsten light (Fig. 14A), however, only the cells within
zoogloeae were apparent by ultraviolet illumination (Fig. 14B) .
Although most of the cells in certain natural finger-like zoogloeae
reacted with the antiserium, relatively few of these were seen in
serum treated activated sludge floes (Fig. 15). However, it may be
that the serum failed to effectively penetrate the floes and make con-
tact with many susceptible cells. In certain cases, activated sludge
floes which did not initiate formation of finger-like zoogloeae did
contain single cells and small clumps of cells which reacted with the
labeled antiserum (Fig. 16).
Certain natural, finger-like zoogloeae did not react with Z_. ramigera
106 labeled antiserum. However, it was noted that when antiserum
positively reacted with a finger-like zoogloea, all visible cells in
the zoogloea were involved in the reaction. In contrast, zoogloeae
not reactive with labeled antiserum contained no fluorescing cells by
ultraviolet light. There were a large number of zoogloeae which did
not react with the labeled antiserum. For example, in slide tests with
zoogloeae developed in scums formed at the surface of mixed liquors
collected from the State College and University Park wastewater treat-
ment plants, only 30 and 54 percent, respectively, of the zoogloeae
counted demonstrated a reaction with Z. ramigera 106 labeled antiserum
(Table 3).
ZOOGLOEAL MATRIX (EXOPOLYMER)
Exopolymer of Zoogloea MP6 was found to be soluble in 0.1 N NaOH but
not in 0.1 N HC1 or lipid solvents (chloroform, acetone, and ethanol).
Bacterial cells of zoogloeae which had been treated with 0.1 N NaOH
appeared largely distorted and it was feared that the intracellular
contents of damaged bacteria might seriously contaminate the exopolymer.
Therefore, milder polymer recovery methods were desired and it was
found that mechanical blending of zoogloeae in 0.02 M K2HP04 or distilled
water freed portions of the polymer while leaving the bacteria, unaffected
(Table 4).
26
-------
Table 2. Specificity of Zoogloea ramigera 106 Antiserum in Fluorescent Antibody
NJ
Tests with Axenic Bacteria
Bacteria
a
Zoogloea strains (direct isolates)
Zoogloea strains (indirect isolates)
Zoogloea ramigera 115
Zoogloea ramigera I-16-M
Streptococcus faecalis
Staphlococcus aureus
Escherichia coli
Proteus vulgaris
Pseudomonas aeruginosa
Pseudomonas fluorescens
Pseudomonas putida
Unidentified isolates
Number of
strains
8
34
1
1
1
1
1
1
3
1
1
71
Number of strains
showing intense
fluorescence
5
3
Oc
0
0
0
0
0
0
0
0
0
Percent of strains
showing intense
fluorescence
63
9
0
0
0
0
0
0
0
0
0
0
Zoogloea strains isolated by Unz and Dondero (65).
Zoogloea strains isolated by Unz and Farrah (68).
Slight initial fluorescence was observed which was eliminated by absorbing conjugated Zoogloea
ramigera 106 antiserum with _Z. ramigera 115.
Bacteria isolated from mixed liquor and the scum formed at the surface of mixed liquor stored
in beakers at 28C for 48 hrs.
-------
Fig. 13. Natural, Finger-Like, Bacterial Zoogloeae Treated with
Zoogloea ramigera 106 Conjugated Antiserum. Specimen obtained
from scum layer which developed on the surface of mixed
liquor stored in a beaker; 48 hr, 28 C. Darkfield condenser
with: A, tungsten light; B, ultraviolet light. Bar equals 10 Mm.
28
-------
Fig. 14. Natural, Finger-Like, Bacterial Zoogloeae and Filamentous
Bacteria Treated with Zoogloea ramigera 106 Conjugated
Antiserum. Specimen obtained from scum layer which developed
on the surface of mixed liquor stored in a beaker; 48 hr,
28 C. Darkfield condenser with: A, tungsten light; B,
ultraviolet light. Bar equals 10 ym.
29
-------
Fig. 15. Bifurcate, Finger-Like, Bacterial Zoogloea and an Activated
Sludge Floe Treated with Zoogloea ramigera 106 Conjugated
Antiserum. Specimen obtained from scum layer which developed
on the surface of mixed liquor stored in a beaker; 72 hr,
28 C. Darkfield condenser with: A, tungsten light; B,
ultraviolet light. Bar equals 10 ym.
30
-------
Fig. 16. Activated Sludge Floes Treated with Zoogloea ramigera 106
Conjugated Antiserum. Brightly fluorescing individual cells
and cell aggregates, presumably Zoogloea sp., are distinguish-
able in floes. Specimen obtained from scum layer which
developed on the surface of mixed liquor stored in a beaker;
72 hr, 28 C. Darkfield condenser with: A, tungsten light;
B, ultraviolet light. Bar equals 10 pm.
31
-------
Table 3. Specificity of Zoogloea ramigera 106 Antiserum in Fluorescent
Antibody Tests with Natural, Finger-like Zoogloeae Present in
Mixed Liquor Scum-3
Source of
mixed liquor
Number of
finger-like
zoogloeae
observed
Number of
finger-like
zoogloeae show-
ing fluorescence
Percentage of
finger-like
zoogloeae show-
ing fluorescence
State College, Pa.,
wastewater treatment
plant 23
University Park, Pa.,
wastewater treatment 110
plant
54
30
49
Scums formed at the surface of mixed liquor stored in beakers in
48 hrs at 28C.
Table 4. Total and Viable Numbers of Bacteria Released from Zoogloea
MP6 Zoogloeae with Dipotassium Phosphate
Molar concentration of
dipotassium phosphate
Viable cell count
per ml x 10~7 b
Total cell count
per ml x 10"'
0.00
0.02
0.05
0.10
42
42
21
0.15
115
105
66
34
Zoogloeae blended for 1 min in solutions; final pH 10.
Mean of duplicate cell counts.
32
-------
Polymer obtained from zoogloeae blended in 0.02 M K2HPOA was s°l-uble and
could be concentrated to form a clear, viscous solution. In contrast,
zoogloeae blended in distilled water released polymer which, upon con-
centration, produced a cloudy, viscous liquid. Exopolymer suspended in
water was cell-free and intact. Zoogloeae of Zoogloea MP6 and cell-free
isolated polymer are shown in Figures 17 and 18.
The efficiency of different treatment methods for polymer recovery varied.
Although blending zoogloeae in water or 0.02 M lUHPO, did not visibly
affect the viability of bacterial cells, less than half of the available
exopolymer could be recovered(Table 5). Boiling zoogloeae in 0.02
M K2HPO^ or approx. O.OOrNNaOH (both solutions having a pH of 10)
allowed separation of most of the exopolymer. For routine analytical
work, exopolymer was obtained by boiling zoogloeae suspensions in 0.02
M K^HPO, for 10 min at pH 10. No amino sugar-containing exopolymers
could be obtained from floes of Z_. ramigera 115 or Z_. ramigera I-16-M
using the aforementioned procedure and the failure to detect amino sugar
in concentrates was considered proof that amino sugars were not being
released from cell walls during treatment (Table 6).
Purified and dissolved Zoogloea MP6 expolymer revealed no absorption
peaks at 260 nm or 280 nm indicating the absence of nucleic acids and
protein (Fig. 39). Hydrolyzed Zoogloea MP6 exopolymer was found by paper
chromatography to consist of two major and one minor chemical components
all of which were reducing and ninhydrin positive (Fig. 20). In an
attempt to identify these compounds, several known reference chemicals
were chromatographed with the hydrolyzed sample (Fig.21). One unknown
substance (spot B) co-chromatographed with D-glucosamine • HC1. Unknown
compound (spot A) migrated similarily to D-glucose, however, it was
unlike glucose in that it gave a positive ninhydrin reaction. Unknown
compound (spot C) was immobile relative to reference and other unknown
chemical components.
Purified Z_. ramigera 106 exopolymer was found by paper chromatography
to be similar in chemical constituents to the polymer of Zoogloea MP6.
Culturing the Zoogloea strains on media of different chemical composi-
tion did not appear to affect the chemical quality of the exopolymer
produced (Table 7).
Evidence of amino sugars in the exopolymers of Zoogloea strains was of
interest regarding the possibility of indirectly quantifying polymer
production by analyzing for amino sugars. The optimum hydrolysis time
for liberation of the amino sugar from exopolymer with boiling aqueous
6 N HC1 was found to be 0.75 hr (Table 8). A longer heating period did
not produce an appreciable increase in free amino sugar.
33
-------
Fig. 17. Zoogloeae of Zoogloea MP6. Zoogloeae treated with India ink
to accentuate matrix. Static culture, sodium lactate-mineral
salts medium; 48 hr, 28 C. Phase contrast; wet mount. Bar
equals 10 ym.
34
-------
Fig. 18. Water-Sheared, Cell-Free Exopolymer of Zoogloea MP6.
Exopolymer treated with India ink to accentuate boundary.
Phase contrast; wet mount. Bar equals 10 ym.
35
-------
Table 5. Separation and Recovery of Exopolymer from Zoogloea MP6.
Exopolymer extraction method
Amino sugar in exopolymer
recovered (yg/mg dry
weight of culture)
Amino sugar in unrecovered
exopolymer (yg/mg dry
weight of culture)3
Blending with 0.02 M
K2HP04, pH 10
Blending with distilled water
Boiling with 0.02 M
K2HP04, pH 10
Boiling with distilled water
adjusted to pH 10 with NaOH
Control (untreated sample)
35
30
70
70
0
45
55
10
15
90
Unseparated exopolymer together with bacterial cells was hydrolyzed in 6 N HC1 for
45 minute in a boiling water bath. Insoluble residues were removed by centrifugation
before the clear supernatant was dried to remove HC1 and analyzed for amino sugar.
-------
Table 6. Separation of Recovery of Exopolymer from Zoogloea ramigera 115 and
Z_. ramigera I-16-M
Zoogloea strain
115
115
I-16-M
I-16-M
Exopolymer
recovery method
Boiling for 10 min
Blending for 1 min
Boiling for 10 min
Blending for 1 min
Amino sugar in
exopolymer recovered t
yg/mg dry weight of culture
0
0
less than 0.1
less than 0.1
Reducing substances
exopolymer recovered
.yg/mg dry weight of
1
0.5
0.5
0.3
in
»
culture
Zoogloeae suspended in 0.02 M
at pH 10.
-------
0.50
220
240. 260 280
Wavelength in nm
300
Fig. 19. Absorption Spectrum of Unhydrolyzed Zoogloea MP6 Exopolymer.
38
-------
B
1
Fig. 20. Comparative Paper Chromatography of Acid Hydrolyzed Zoogloea
MP6 Exopolymer Revealing Spots A, B, and C. Isopropanol-
water (4:1) solvent system. Chromatogram number 1 processed
with ninhydrin. Chromatogram number 2 processed with silver
nitrate reagent.
39
-------
Fig. 21. Paper Chromatography of Acid Hydrolyzed Zoogloea MP6 Exo-
polymer and Reference Compounds. Isopropanol-water (4:1)
solvent system. 1, D-glucuronic acid; 2, D-glucosamine-HCl;
3, exopolymer hydrolysate; 4, muramic acid^HCl; 5, D-glucose.
Chromatogram processed with silver nitrate reagent.
40
-------
Table 7. Paper Chromatography of Acid Hydrolyzed Exopolymer Obtained
from Zoogloea ramigera 106 and Zoogloea MP6a
RD-glucosamine • HC1
values of exopolymer components
Strain numb
106
MP6
MP6
MP6
er Culture medium Spot A Spot B
Basal medium + sodium
lactate 1.25 1.00
Basal medium + sodium
lactate 1.27 1.00
Casitone - yeast
autolysate 1.40 1.08
Trypticase Soy 1.30 1.03
Spot C
0.07
0.14
0.07
0.07
a
Solvent system: isopropanol - water (4:1)
Table 8. Free Amino Sugar Content of Zoogloea MP6 and Zoogloea
ramigera 106 Exopolymers Hydrolyzed with 6 N HC1 in Boiling
Water
Hydrolysis
period
(hr)
0.25
0.75
2.0
6.0
12.0
Percent of
sugar released
Strain MP6
91
95
100
91
91
maximum amino
from exopolymer
Strain 106
100
96
96
94
89
41
-------
Elution of the major chemical components from hydrolyzed Zoogloea MP6
exopolymer was accomplished by column separation. However, only two
substances were recovered having R glucosamine values of 0.95 and 1.77,
respectively (Fig. 22). The substance designated spot C on paper
chromatograms traveled very poorly and was not recovered- In all prob-
ability, the compound which appeared first in the column effluent was
glucosamine and corresponds to spot B on paper chromatograms. The
second amino sugar to leave the column may have been fucosamine although
no reference standard was available for comparison.
Hexosamines were not found to exist as the N-acetyl form in crude
Zoogloea MP6 zoogloeae or in purified and hydrolyzed Zoogloea MP6
exopolymer or water suspensions of cell free exopolymer (Table 9).
However, amino sugars were present in all of these samples.
Quantitative analyses of Zoogloea MP6 exopolymer revealed the dry weight
of acid hydrolyzable fraction to be 69 percent of polymer dry weight
with reducing substances and amino sugars accounting for approximately
19 and 16-18 percent, respectively, of the polymer dry weight. Hexose,
uronic acids, and ether soluble substances constituted approximately
one percent of the dry weight of polymer (Table 10).
Purified and unhydrolyzed Zoogloea MP6 exopolymer was titrated with
0.01 N NaOH and an equivalent weight of approximately 1500 was obtained
which corresponds to one acidic residue per 10 carbohydrate residues.
The exopolymers of natural zoogloeae and Zoogloea MP6 were not observed
to be microfibrilar by electron microscopy.
Chemical analyses of acid hydrolyzed activated sludge exopolymer
revealed hexose and amino sugars but no uronic acids. Amino sugars were
the major portion of the reducing substances liberated (Table 11).
However, the percentage of amino sugars and reducing substances
liberated were much lower than that found upon analysis of the exopolymer
of Zoogloea MP6. Paper chromatography of the acid hydrolyzed activated
sludge exopolymer revealed two major reducing spots, one of which could
be identified as glucosamine. On this basis, activated sludge polymer
bears some resemblance to the exopolymer of Zoogloea MP6, however,
column chromatography and fractionation of the sludge polymer is needed
to provide a more meaningful analysis of the chemical composition.
42
-------
20
-P-
Co
I 15
0)
o
u
O
10
0)
oo
en
o
-S
I
60
D-Glucosamine-HCl
O Hydrolyzed Zoogloea MP6 Exopolymer
100
Tube Number
150
Fig. 22. Column Separation of Amino Sugars Present in Acid Hydrolyzed Zoogloea MP6
Exopolymer. Elution by 0.3 N HCl on Dowex 50 column.
-------
Table 9. Absence of N-Acetyl Hexgsamines in Exopolymer of Zoogloea MP6
Ug N-acetyl ^g amino sugar
hexosamines liberated from
(N-acetyl glucosamine acid hydrolyzed
Nature of samples used as standard) samples'3
Suspension of zoogloeae
•a
Exopolymer dissolved with K~HPO,
a
Exopolymer removed with water
0
0
0
160
40
115
a
Samples homogenized for 1 minute in a Waring blender; cells removed
by centrifugation at 27,000 x gfor 10 minutes
Samples hydrolyzed with 6 N HCl for 45 minutes in a boiling water
bath; HCl removed by drying samples in air.
44
-------
Table 10. Chemical Composition of Zoogloea MP6 Exopolymer'
Weight of
substance
Percent of
unhydrolyzed
Analytical test
Dry weight, unhydrolyzed sample
Reducing substances
Amino sugar
Hexose
Uronic acid
Reference
compound
D-glucosamine'HCl
D-glucosamine-HCl
D- glucose
D-glucuronic acid
(mg)
Run 1
3.2
0.61
0.53
0.03
0.03
Run 2
1.0
0.19
0.18
0.03
0.01
sample
Run 1 Run 2
100
19
16
1
1
100
19
18
3
1
Cells cultured in sodium lactate - mineral salts medium; exopolymer removed from cells
by blending with 0.02 M K HPO,, pH 10, and purified by precipitation with
cetyltrimethylammonium bromide.
-------
Table 11. Chemical Composition of Activated Sludge Exopolymer
Sample
Analytical
test
Weight of
substance
recovered (rag)
Percent by weight
of unhydrolyzed
sample
Unhydrolyzed polymer Dry 78.0
weight
Hydrolyzed polymer Amino 1.0
sugar
Reducing 1.5
substance
Hexose 2.9
Uronic 0.0
acid
100.0
1.3
1.9
3.7
0.0
46
-------
BACTERIAL FLOCCULATION
Growth and zoogloea formation by_ Zoogloea MP6
The growth cycle of Zoogloea MP6 cultured in sodium lactate-mineral salts
medium is shown in Figure 23. An index of flocculation (I. F.) equal to
the difference between optical density values of unsettled (homogenous)
and 4-hr settled culture divided by the optical density value of the
unsettled culture was used to compare the flocculation of organisms at
different stages of the growth cycle and under different culture con-
ditions. Large I. F. values were indicative of good bioflocculation
and values of 0.20 and less were considered representative of non-
flocculent cultures. Measurable flocculation of Zoogloea strains was
found to begin in the late logarithmic phase of growth becoming
maximum at the onset of the stationary phase. During early stages of
flocculation, a few encapsulated cells were observed to form a nucleus
to which other bacteria attached themselves (Fig. 24). Bacterial
flocculation proceeds at a more rapid rate than amino sugar production
although amino sugars continued to be synthesized and incorporated into
exopolymer even after maximum bacterial flocculation was reached.
Apparently, exopolymer production does not cease even when the'net
growth approaches zero.
Metal ions and flocculation
Metallic cations, including mono-, bi-, and tri-valent forms, had little
effect on promoting flocculation of Zoogloea MP6 (Figs. 25 and 26).
Zinc and manganese salts were toxic in concentrations of 0.01 mM and
1.0 mM, respectively. Only calcium salts in concentrations of 0.1 mM
or greater appreciably influenced flocculation of the bacteria as
evidenced by large I. F. values. However, calcium ion was not always
effective as a flocculating agent for the Zoogloea strains tested. On
one occasion, Z_. ramigera 106 cells did not flocculate well in the
absence or presence of calcium ion (Fig. 27). Judging from the results
of the foregoing experiments, it appears that calcium ion merely serves
to increase the efficiency of cell aggregation over that possible
through natural bioflocculation and does not induce normally dispersed
cells to coalesce. Index of flocculation values for Zoogloea MP6, 21,
and "L. ramigera 106 cultured in the presence of various concentrations
of metal ions are presented in Table 12. Both NaBr and NaF were tested at
Na ion concentrations of 1.0 millimole per liter and produced no effect
on flocculation. The variability experienced in the effect of calcium
ion on flocculation of Zoogloea strains prompted further investigation
into the nature of the test Organisms. It was noted that the extent of
flocculation varied from time to time with the same Zoogloea strain
under similar culture conditions. Although cultures of well and poorly
floc'-'ildced cells both formed surface films on standing, these pellicles
did not resist agitation equally well. Mechanical shaking of the well
flocculated culture disrupted the pellicle into small fragments although
cells remained in zoogloeae and the supernatant was practically clear.
47
-------
Viable Count
Optical Density
Amino Sugar/Dry Weight of Cell Mass
A A Flocculation Index
12r 0.60r
0 10 20 30 40 50 60 70
Hours
Fig. 23. Growth, Flocculation, and Amino Sugar Production by Zoogloea
MP6. Sodium lactate-mineral salts culture medium.
-------
Fig. 24. Development of Zoogloeal Floes by Zoogloea MP6. A, nucleus
of zoogloea-forming cells in tiny floe; B, larger floe con-
sisting of nucleus of zoogloea-forming cells to which many
nonencapsulated bacteria are attached. Floes treated with
India ink to accentuate zoogloeal matrix boundary. Sodium
lactate-mineral salts culture medium; 32 hr, 20 C. Phase
contrast; wet mount. Bar equals 10 ]Jm.
49
-------
Optical Density (Unsettled Culture)
0.8 ,-
O Optical Density (Settled Culture)
0.0
0.001
ZnSO,
0.01
Cations, mM
0.1
1.0
Fig. 25. Influence of Cations on Flocculation of Zoogloea MP6,
50
-------
00
Ui
n
o
ft
ft
0.8
0.4
§ 0.2
cn
C
0)
Q
cfl
O
•H
4-1
0.0
0.8
CaSO,
0.4
0.2
0.0
0 ;
• Optical Density (Unsettled Culture)
O Optical Density (Settled Culture)
_L
0.001
I
0.01
Cations, mH
0.1
1.0
-------
Optical Density (Unsettled Culture)
-O Optical Density (Settled Culture)
1.0 r-
0.8 U
1.00
Fig. 26. Influence of Magnesium Ion on Flocculation of Zoogloea MP6
and Zoogloea ramigera 106.
52
-------
Optical Density (Unsettled Culture)
Optical Density (Settled Culture)
(-0
0.001
0,01
CaSO mM
0.1
1.0
Fig. 27. Influence of Calcium Ion on Flocculation of Zoogloea ramigera 106.
-------
Table 12. Influence of Metal Ions on Flocculation of Zoogloea MP6,
21 and Z_. ramigera 106
Zoogloea
strain no. Cation
MP6 Ca"1"1"
MP6 Mg"^
MP6 Mn4"1"
MP6 Zn**
MP6 Na+
MP6 Fe"H~t'
106 Ca*4"
Cation
concentration Index of Flocculation
(millimole/1) Experiment 1 Experiment 2
1.0
0.1
0.01
0.001
0.0
1.0
0.1
0.01
0.0
1.0
0.1
0.01
0.001
0.0
1.0
0.1
0.01
0.001
0.0
1.0
0.1
0.01
0.001
0.0
1.0
0.1
0.01
0.001
0.0
1.0
0.1
0.01
0.001
0.0
0.71
0.47
0.18
0.16
0.14
0.06
0.04
0.08
0.14
c
0.11
0.04
0.05
0.14
—
—
0.01
0.14
0.05
0.06
0.10
0.07
0.14
(salt precipitated)
0.18
0.20
0.19
0.14
0.25
0.22
0.18
0.17
0.19
0.72
NTd
NT
NT
0.36
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0.72
NT
NT
NT
0.35
54
-------
Table 12, (Cont'd)
Cation
Zoogloea concentration
strain no. Cation3 (millimole/1)
106 Mg"1"4" 1,0
0.1
0.01
OoO
21 Ca44" 1.0
0.0
Index of Flocculation
Experiment 1 Experiment 2
0,0
0.04
0.20
0.19
0.21
0.09
NT
NT
NT
NT
NT
NT
o
All cations present as compounds of sulfate
Index of Flocculation (I» F.) values determined from optical density
measurements on settled and unsettled cultures after 48 hr incubation
using the formula: I« F, = 00D850Q Unsettled-O.D.Settled
O.D.5QQ Unsettled
— indicates cation was toxic
NT indicates cation was not tested
55
-------
However, similar agitation of nonflocculating cultures resulted in a
dispersion of the cells in the surface film making the culture fluid
strikingly turbid. Spread plating serial dilutions of each type of
culture gave rise to two colonial forms. The "rough" colony was
raised with a rugose surface whereas the "smooth" colony was slightly
raised with a smooth surface (Fig. 28). Rough colony forming bacteria
were present in major proportions in flocculating cultures of Zoogloea
MP6 and were only minor in nonflocculating cultures (Table 13). When
separate cultures developed from single rough and smooth colonies were
agitated on the gyrotary shaking machine, only the rough type culture
demonstrated flocculation (Table 14).
Temperature and flocculation
Zoogloea MP6 increased rapidly in cell mass when cultured on trypticase
soy broth at 28 C and 36 C and more slowly at 20 C. Bacterial zoogloeae
were formed at all temperatures. Exopolymer production, based on amino
sugar analysis of purified zoogloea matrix, paralleled the increase in
dry weight (Fig. 29) and it could not be determined that polymer pro-
duction was especially favored at any of the temperatures tested.
Reducing agents and flocculation
In separate trials, various concentrations of sodium thioglycollate,
sodium ascorbate, and sodium thiocyanate were incorporated in agar
underlayers in beakers containing lactate-mineral salts medium
inoculated with Zoogloea MP6. Beakers were kept well covered to avoid
contamination during incubation. Both sodium thioglycollate (Fig. 30)
and sodium ascorbate (Fig. 31) were effective in depressing the
oxidation-reduction potential and a zoogloeal film developed at the
surface of the medium. The amino sugar content per unit of film dry
weight increased at higher concentrations of sodium thioglycollate.
Optical density values of culture fluids were lower at the higher
concentrations of sodium thioglycollate and sodium ascorbate indicat-
ing a low density of dispersed bacteria in the culture medium below
the surface film. Sodium thiocyanate did not effect lowering of the
oxidation-reduction potential as did the reducing agents and the dry
weight of the surface film was less at any concentration of sodium
thiocyanate used than in control cultures (sodium thiocyanate absent).
However, amino sugar per unit weight of film did increase with increas-
ing concentrations of sodium thiocyanate (Fig. 32). In all cases,
increased concentrations of any of the three sodium salts in the agar
underlayer resulted in cells becoming stratified closer to the surface
of the culture medium indicating, possibly, that a toxicity gradient
existed in the supernatant. As such, toxicity of the chemical agents
rather than oxidation-reduction potential may have been the influential
factor in determining the thickness of the surface film formed and the
vertical range in the liquid medium within which cell proliferation
occurred.
56
-------
Fig. 28. Colonies of Zoogloea MP6 on Solid Culture Medium. A, smooth; B, rough.
Sodium lactate-mineral salts culture medium; 5 days, 28 C. Photographed by
reflected light. Bar equals 1.0 mm,
-------
Table 13. Flocculation and Colonial Morphology (Solid Culture Medium) of Zoogloea MP6
Optical Density Number of Number of
of 48 hr culture Index of colonies rough colonies
Flask No.a Initial Settled Flocculation^ examined observed
1 0.49 0.25 0.49 232 216
2 0.46 0.43 0.07 155 38
Eercent
i rough
colonies
93
24
Ul Q
<» Growth medium was the basal medium (BM) with sodium lactate (1.0 g/1) , culture flasks
incubated at 20C on gyrotary shaker.
Index of Flocculation (I. F.) values determined from optical density measurements on
settled and unsettled cultures after 48 hr incubation using the formula:
I. F. = 0'I>.500 Unsettled .-. O.D.50Q Settled
O.D.5 Q Unsettled
-------
Table 14. Flocculation of Rough and Smooth Cultures of Zoogloea MP6'
Optical density
Culture
type
Rough
Smooth
of 48 hr
Unsettled
0.45
0.56
culture
Settled
0.22
0.51
Index of Flocculation
0.51
0.09
Growth medium was the basal medium (BM) with sodium lactate (1.0 g/1);
culture flasks incubated at 20 C on a gyrotary shaker
Index of Flocculation (I.F.) values determined from optical density
measurements on settled and unsettled cultures after 48 hr incubation
using the formula: O.D. Unsettled - O.D. Settled
Unsettled
-------
ON
O
18
16
Q)
14
3
a
12
03
W
10
<" o
o 8
M-l
O
60
•H
1-1
n
400
0)
£ 300
3
cd
60
o 200
G
60
100
I
I
-A 20 C
-O 28 C
-• 36 C
20
40 60
Hours
80
100
20
40 60
Hours
80
100
Fig. 29. Growth and Amino Sugar Production by Zoogloea MP6 at Different
Incubation Temperatures. Static culture, trypticase-soy broth.
-------
100 r-
0.30
g 0.20
CD
C
0)
O
0.10 -
cfl
O
•H
•u
ex
O
0.00
Amino Sugar/Cell Mass
-• Oxidation-Reduction Potential
-A Optical Density
Cell Mass
400
0.01 0,10
Sodium Thioglycolate (mM)
1.0
4-1
iH
3
O
60 .
500
300 I.
efl
•H
4J
C
CD
to
to
- 40
Fig. 30. Influence of Sodium Thioglycollate on Amino Sugar Production by Zoogloea MP6.
-------
100 r-
0.3
o
o
•H
CO
£
0)
P
3
P.
o
0.2
0.0
CO
CD
Cfl
a
tH
0)
o
80
60
60
M
n)
oo
40
8
•H
I
60
20
-,500
Amino Sugar/Cell Mass
Oxidation-Reduction Potential
Optical Density
D Cell Mass
I
400
300
c
0)
4J
O
200 g
T)
ai
100
c
o
•H
•U
ta
0.01 0.1
Sodium Ascorbate (mM)
1.0
III
n
4J
r-\
3
O
CO
CO
CU
O
M-l
O
60
20
10
Fig. 31. Influence of Sodium Ascorbate on Amino Sugar Production by Zoogloea MP6,
-------
100
GJ
0.3
I
o
o
LT|
CO
C
cfl
O
•H
4-1
P.
O
0.1
0.0
80
ca
M
•8 60
fi
60
•H
40
60
cd
60
3
C/3
§ 20
60
-O Amino Sugar/Cell Mass
-• Oxidation-Reduction Potential
-A Optical Density
-D Cell Mass
_500
.300
400
200
4-1
C
CO
4->
O
PL.
C
O
CJ
3
100
tfl
3
x
o
0.01 0,10
Sodium Thiocyanate (tnM)
1.0
4-1
rH
CJ
00
e
CO
CO
20
10
43
60
Fig. 32. Influence of Sodium Thiocyanate on Amino Sugar Production by Zoogloea MP6.
-------
Carbon _to_ nitrogen ratios and flocculation
Cells of Zoogloea MP6 flocculated well at nitrogen concentrations which
did not limit the extent of growth in respect to the available carbon
(substrate). At nitrogen concentrations which prevented maximum biomass
production, index of flocculation values decreased considerably (Table 15)
Flocculation could not be improved by increasing the nitrogen concentra-
tion of the culture medium above that required to attain maximum biomass.
Decreased flocculation at low nitrogen concentrations may be related to
reduced exopolymer production by the bacteria. At least the amino sugar
to dry weight of culture ratios were much lower under these conditions
than when higher concentrations of nitrogen were available in the culture
medium. Varying the carbon content of the culture medium when provid-
ing a high initial nitrogen concentration did not affect the amino sugar
production and index of flocculation values were consistently high.
NATURAL BACTERIAL ZOOGLOEA FORMATION
Mixed liquors stored in beakers for 48 hr at 28 C underwent changes in
chemical and bacteriological characteristics, primarily: (a) lowering
of the oxidation-reduction potentials (b) increase in volatile acid
content, and (c) development of a scum with natural zoogloeae at the
surface. The extent to which these effects developed seemed influenced
by the mixed liquor suspended solids (MLSS) content of the mixed liquor,
strength of the wastewater upon which the solids were produced or a.com-
bination of these. Mixed liquor obtained from the State College
biooxidation basin, which receives a strong, unsettled domestic waste-
water, exhibited a sharply lowered oxidation-reduction potential and an
increased volatile acid concentration during storage. Increasing the
MLSS content of the mixed liquor in beakers resulted in an increased
rate of change both in oxidation-reduction potential and volatile acid
content (Fig. 33A). The scum which formed at the surface of the
mixed liquor contained exopolymer produced by the zoogloea-forming
bacteria and it was found that both amino sugar and dry weight of scum
also increased when the mixed liquor had been supplemented with additional
MLSS prior to storage (Fig. 33B). Mixed liquor obtained from the
University Park activated sludge aeration basin, which receives a moderate
strength primary effluent, remained essentially unchanged during storage
unless the solids content of the mixed liquor was significantly increased
prior to incubation in beakers (Fig. 34). The mixed liquor storage
experiments were repeated using fresh samples of mixed liquor collected
from the State College and University wastewater treatment plants and
the results confirmed earlier findings, all of which are summarized in
Table 16. It should be noted that the mixed liquor of the State College
secondary biooxidation basin, which treats the effluent from the primary
biooxidation unit, displayed no change during storage and it was not
possible to initiate scum formation by increasing the solids content.
Occasionally, however, small fragments of MLSS would collect at the
surface of mixed liquor during storage and be inadvertently included in
dry weight of scum determinations.
64
-------
Oi
Table 15. Influence of Carbon to Nitrogen Ratio on Flocculation and Amino Sugar
Production by Zoogloea MP6
Carbon as
sodium
lactate (g/1)
0.32
0.32
0.32
0.32
0.32
0.32
1.28
0.64
0.16
0.08
Nitrogen
as ammonium
sulfate (g/1)
0.55
0.28
0.11
0.028
0.011
0.0028
0.056
0.056
0.056
0.056
Ratio of
carbon to
nitrogen
0.6
1.1
2.9
11.4
29.0
114,0
24.0
12.0
3.0
1.5
Index of
Flocculation
0.50
0.48
0.62
0.68
0.31
0.03
0.42
0.50
0.45
0.33
Dry weight
of culture
(mg/50 ml
of culture
fluid)
10.9
10.8
13.2
9.3
5.1
1.7
12.1
5.6
1.4
0.6
Amino
sugar
(yg/50 ml.
of culture
fluid)
440
390
460
320
80
20
580
280
80
30
Ratio of
yg amino
sugar/ml
dry weight
of culture
41
36
35
35
16
12
48
50
57
53
Index of Flocculation (I. F.) values determined from optical density measurements on
settled and unsettled cultures after 48 hr incubation using the formula:
I. F. = O.D.,
'500
Unsettled - O.D.5QO Settled
O.D.5Q Unsettled
-------
B
4J
0)
c
O
•H
4J
O
3
I
O
•H
4-1
Cfl
T)
O 620 mg of Solids
• 460 mg of Solids
— Oxidation Reduction Potential
•— Volatile Acid
400
300-
200-
100-
O 620 mg of Solids
• 460 mg of Solids
Dry Weight of Scum
Amino Sugar
j_
50 3
a
40 3!
v—'
30 S
60
20^
o
101
0 10 20 30 40 50 60 70
Hours
0 10 20 30 40 50 60 70
Hours
Fig0 33. Chemical and Microbiological Characteristics of State College Mixed
Liquor During Storage, A, oxidation-reduction potential and volatile
acids; B, dry weight of surface scum and amino sugar content,,
-------
B
500
400
c
cu
| 300
g
4J
O
13
200
•H 100
n)
13
•H
• 380 mg of Solids
O 630 mg of Solids
Oxidation Reduction
Potential
Volatile Acids
25
\
20
0 10 20 30 40 50 60 70
Hours
• 380 mg of Solids
O 630 mg of Solids
Dry Weight of Scum
Amino Sugar
a a)
•H M
4J 3
O rH
O
0
0)
(-1
3
4J
,-t
O
60
15
,0
5
0
00
0 10 20 30 40 50 60 70
Hours
Fig. 34. Chemical and Microbiological Characteristics of University Park
Mixed Liquor During Storage. A, oxidation-reduction potential and
volatile acids; B, dry weight of surface scum and amino sugar content.
-------
Table 16. Physical, Chemical, and Microbiological Characteristics of Stored Mixed Liquor0
oo
Source of
mixed
liquor and
sample
letter
State College
Aeration
tank 1 -A
-B
tank 2 -A
-B
University Park
Aeration
tank -A
-B
C.O.D.
of
influent
wastewater
(mg 02/1)
500
500
40
40
180
180
Volume
of
mixed
liquor
stored
(ml)
200
200
200
200
200
200
Volume
of
mixed
liquor
suspended
solids
stored
(ml)
51
85
47
68
43
71
Weight
of
solids
stored
(mg dry
weight
510
850
376
545
382
632
Oxidation- reduction
potential of
stored
mixed liquor
(mv)
Initial
+490
+490
+490
+490
+485
+485
Final
-31
-44
+449
+410
+407
+177
Organic
a.cids
(mg/1
as
acetic
acid)
22
39
0
0
0
27
Microbial
mg dry Ug
scum
amino
weight sugar
6.6
11.0
0.8
0.8
0.0
1.0
136
220
0
0
0
11
Mixed liquor stored in beakers for 72 hr at 28 C.
-------
Since the appearance of a zoogloea-containing scum was regularly accom-
panied by increased volatile acids in the mixed liquor and lowered
oxidation-reduction potential, additional studies were conducted to
resolve the influence of each of these variables on scum formation.
Agar containing a reducing agent was allowed to solidify at the bottom
of a beaker before adding a volume of mixed liquor. The use of reduc-
ing agents alone to rapidly lower the oxidation-reduction potential did
not promote scum formation in stored University Park mixed liquor.
However, addition of sodium lactate to the mixed liquor prior to incu-
bation regardless of whether or not a reducing agent was included,
resulted in appreciable scum development which was measured in terms of
dry weight and amino sugar content (Table 17). Oxidation-reduction
potential was observed to decrease naturally with time in the absence
of a supplementary reducing agent. In these experiments, scum formation
took place only when sodium lactate was initially added to mixed liquor
and was unrelated to the final value of the oxidation-reduction potential.
Eight substrates, in addition to sodium lactate, were tested for their
ability to support scum formation in stored mixed liquor. Scums were
harvested from each nutrient-enriched mixed liquor after 72 hr storage
and placed in test tubes (Fig. 35). The dry weight and amino sugar
content of the scum substance was determined (Table 18). Starch,
glucose, sodium lactate, and sodium m-toluate visibly supported heaviest
scum formation although, on the basis of amino sugar content, polymer
production was enhanced by all nutrients except sodium p_-toluate.
Amino sugar generally increased with the dry weight of scum produced
although the ratios of amino sugar to dry weight varied depending on
the nutrient supplied. This variation may be expected since, in some
cases, unavoidable inclusion of small portions of buoyant MLSS with
harvested scum exaggerated scum dry weight values. All scums were
heavily populated with finger-like zoogloeae. Although it was surpris-
ing to find that substrates such as starch and glucose supported
relatively heavy development of finger-like zoogloeae, it may be that
thse nutrients were decomposed by other micoorganisms in the mixed
culture and that the zoogloeal bacteria utilized the metabolic depot
products of nonzoogloeal organisms.
69
-------
Table 17. Influence of Reducing Compounds and Sodium Lactate on the Formation of Scum
at the Surface of Mixed Liquor Stored in Glass Beakers3
Reducing compound
Initial
concentration of
sodium lactate
in the supernatant
/lV
Final
oxidation-reduction
potential
Scum produced
mg dry weight yg amino sugar
None
None
None
Sodium thioglycolate
Sodium thioglycolate
Sodium thiocyanate
Sodium ascorbate
0
50
200
50
0
0
0
+433
+48
-260
-96
+106
+270
-344
0.2
6.4
6.4
2.0
0.2
0.2
0.2
10
126
152
54
10
6
6
Each 600 ml beaker contained 200 ml University Park mixed liquor over 40 ml agar
to which 1.0 millimole of a reducing compound was added.
Analysis performed after 24 hr incubation of the mixed liquor at 28 C.; initial (time
zero) oxidation-reduction potential for all cultures was essentially +432 mv.
-------
Fig. 35. Scum Layers Harvested from Beakers of Stored, Fortified Mixed
Liquor. Nutrients added to mixed liquors: 1, none;
2, D-glucose; 3, starch; 4, D-xylose; 5, sodium lactate;
6, sodium acetate; 7, sodium glutamate; 8, sodium aspartate;
9, sodium £-toluate; 10, sodium m-toluate. Scums harvested
after 72 hr incubation at 28 C. ~"
71
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Table 18. Influence of Organic Compounds on Scum Production at the Surface of Mixed
Liquor Stored in Glass Beakers3
Final
Substrate Concentration oxidation-reduction
added (mg/l)b potential (mv^
Scum produced4^
mg dry weight yg amino sugar
Ug amino sugar/
mg dry weight
of scum
None
o-toluate
starch
glucose
lactate
m-toluate
glutamate
aspartate
acetate
xylose
56
90
90
90
56
108
156
107
90
+128
+200
+97
+67
+38
+60
+83
+75
+80
+127
1.6
1.2
4.3
4.9
5.0
5.0
4.2
2.9
3.9
2.4
34
34
148
126
126
170
136
136
90
80
21
28
34
26
25
34
32
47
23
33
a
Each 400 ml beaker contained University Park mixed liquor (430 mg suspended solids;
200 ml total volume of mixed liquor).
Concentration of substrate added based on theoretical requirement of 3 millimoles
02/1 for complete oxidation of the anion.
Initial (time zero) oxidation-reduction potential for all cultures was essentially +441 HIV.
Harvested after 72 hr incubation at 28 C.
-------
Section VI
DISCUSSION
The property of zoogloea formation is not restricted to any particular
genus of bacteria. Examples of microorganisms capable of developing
zoogloeae are Siderocapsa sp., certain autotrophic nitrifying bacteria,
Azotobacter sp., Thiodendron sp., Thiocystis sp., and certain chemo-
organotrophic pseudomonads, e.g., Zoogloea sp. Distinctive finger-like
and dendritic zoogloeae originally described and assigned the binomial
epithets, Zoogloea ramigera, by Itzigsohn (31) were first photographed
by Koch (38). Koch envisioned that bacterial cells present in micro-
colonies were so aligned that their proliferation gave rise to zoogloeae
of peculiar shapes. Friedman and Dugan (23) contended that fingered
zoogloeae resulted from an arrangement of individual packets of cells.
It is evident from our studies that finger-like zoogloeae develop as a
consequence of the unidirectional movement and multiplication of
zoogloea-forming bacteria which may or may not originate in activated
sludge floes, microbial slimes and films, etc. It may be imagined that
a marked coordination of cellular activities is required to produce
these unusual zoogloeal structures, and, for this reason, it is anti-
cipated that the formation of finger-like zoogloeae is restricted to
the operations of a very few bacterial species. In effect, finger-like
zoogloeae are the result of groups of bacteria able to move in a liquid
environment while maintaining direction and colonial integrity. The
movement of colonies of Bacillus circulans on solid medium is regarded
as a rate activity among procaryotic organisms (39).
Results of fluorescent antibody experiments in which Z_. ramigera 106
antiserum totally reacted with certain natural, finger-like zoogloeae
lends support to our belief that the colonial structures are formed by
bacteria of the genus Zoogloea. That many natural, finger-like
zoogloeae did not react with the strain specific antiserum strengthens
the view of others that several strains of Zoogloea probably exist in
nature (23, 26). The absence of a cross reaction between Z_. ramigera
106 antiserum and mixed cultures of filamentous, sheathed bacteria
resembling Sphaerotilus sp. casts doubt on earlier suggestions that
Z. ramigera is a growth form of Sphaerotilus sp.
Factors specifically responsible for stimulating the formation of
finger-like zoogloeae remain unknown. However, it appears that the
phenomenon involves an aerotactic or chemotactic behavior by the
zoogloea-forming bacteria. The possibilities are that (a) oxygen lim-
itations in floes cause zoogloea-forming bacteria to grow outward in
the direction of a higher oxygen tension, (b) concentrated waste products
of floe bacteria induce zoogloea-forming bacteria to grow away from the
floe and (c) active bacteria outside the floe produce substances which
attract zoogloea-forming bacteria. At the least, it is visibly apparent
that activated sludge floes contain zoogloea-forming bacteria which
73
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respond rapidly to environmental changes. The implications are that
other, less obvious, changes in the activities and composition of the
general sludge microflora may take place in response to altered environ-
mental conditions. Although many studies have been conducted on the
microbial composition of activated sludge (2,5,7,18,35,36,41,45,46,62,
69), little information is available to substantiate whether or not
qualitative and quantitative changes regularly occur among the micro-
organisms in sludges. Some workers (1, 32) have compared the microbial
composition of activated sludges at different stages of metabolism and
age, however, their techniques would not be sensitive enough to reveal
very subtle differences in sludge microflora over short intervals of
time, e.g., semi-daily or less. Development of methods to analyze very
carefully the stability as well as the composition of the microflora
of activated sludge would greatly enrich the knowledge and remove some
of the mystique attendant to biological wastewater treatment systems.
On the basis of our investigations of activated sludge floes using
microculture and fluorescent antibody techniques, it appears that the
activated sludge process is not especially conducive to the formation
of finger-like zoogloeae. As previously noted by Unz and Dondero (65),
these zoogloeal structures are infrequently observed in fresh activated
sludge floes. Possibly, an occurrence of finger-like zoogloeae in
fresh activated sludge reflects a change in wastewater composition or
treatment plant operations. The appearance of zoogloeae at the surface
of standing wastewater effluent is taken to be evidence of insufficient
treatment in the so-called Kolkwitz "zoogloea test" (40).
The property of zoogloea formation may be advantageous to the pertinent
bacteria in (a) protecting cells from predators, (b) maintaining large
numbers of cells in a fixed location thereby assisting domination and
survival of the organisms, (c) allowing adsorption and absorption of
nutrients on the surface of the Zoogloeal matrix which may also act to
transfer soluble substrates and waste products from regions of higher
to lower concentrations and, (d) incorporating other nonzoogloeal but
biochemically active bacteria in the microbial assemblage. Unz and
Dondero (67) isolated several genera of bacteria from natural, finger-
like, wastewater zoogloeae which they found to be much more active in
certain biochemical tests than Zoogloea sp. and suggested that the non-
zoogloeal bacteria in the wastewater environment may act upon accumu-
lated nutrients unavailable to Zoogloea sp.
Several publications have appeared in connection with the chemistry of
the capsules and exopolymers of bacteria alleged to be zoogloeal (5,
13,24,52,60,70). Among these, amino sugars were referred to only three
times. Anderson (5) isolated two monosaccharides from the exopolymer
of a zoogloea-forming bacterium which he designated glucosamine and
arabinose. Crabtree £t_al. (13) obtained hexosamines in hot water
extracts of floes and cells of £. ramigera I-16-M. Most recently,
74
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Tezuka (60) found two amino sugars in the exopolymer of a zoogloeal
isolate and identified the compounds as N-acetyl glucosamine and
possibly, N-acetyl fucosamine. We believe that the two amino sugars
obtained by us in chemical analyses of the exopolymers of Zoogloea MP6
and "L. ramigera 106 are similar to those described by Tezuka except that
we could not determine that the amino sugars were N-acetyl derivatives.
However, unlike Tezuka, we specifically performed a chemical test for
the N-acetyl groups. Nevertheless, our Zoogloea strains compare closely
with the culture of Tezuka in several important cultural and physio-
logical characteristics. Amino sugars appear to be a convenient class
of substances to analyze for in determining indirectly the exopolymer
content of cultures of Zoogloea sp. However, since amino sugars are
only an indirect measure of exopolymer, it must be emphasized that this
parameter will not be valid for all types of exopolymers including
those of certain strains designated Zoogloea.
It is not surprising to find that the reducing substances and amino
sugar content of activated sludge exopolymer is much lower percentage-
wise than in Zoogloea sp. exopolymer. As inferred by Busch and Stumm
(9), sludge exopolymer is probably composed of the extracellular
products of several types of resident bacteria, all of which are unique
in certain characteristics. The presence of noncarbohydrate substances
in the sludge polymer would diminish the amino sugar content of the
polymer on a percentage basis. Others have reported on the existence
of major components of a noncarbohydrate nature in sludges (37,49, 50,
53, 70) and the presence (70) and absence (22,62) of hexosamines and
N-acethylhexosamines.
Our observation that intense flocculation by Zoogloea strains commences
in late logarithmic growth phase confirms earlier results of Finstein
(21). It appears that, depending upon culture conditions, zoogloea
formation may result from a few encapsulated cells which coalesce and
increase the size of the floe by merging with other cells, multiplication
of individual polymer producing bacteria, and a combination of these.
The former mechanism is more likely to occur in agitated cultures where
random collision of cells is frequent. Under quiescent conditions,
zoogloea-forming bacteria would be expected to develop zoogloeae
primarily through multiplication and exopolymer production. We have
repeatedly found that dispersed cells of Zoogloea strains are clearly
encapsulated only during the flocculation phase. Others have commented
on the lack of capsules on cells of zoogloeal bacteria (8,13,26).
Calcium ion has been found stimulatory (59,69), inhibitory (6) and
neutral (58) in connection with the flocculation of microorganisms. In
the present study, calcium either strongly enhanced the flocculation of
cells of Zoogloea strains or had no effect. The nature of the floe
promoting property of calcium ion cannot be described as mere electro-
static attraction since several other bivalent cations did not induce
flocculation under any experimental conditions. Perhaps calcium ion
75
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is involved in chelation with negatively charged grovps on the exopolymer
resulting in a bridge between bacterial cells. Peter and Wuhrmann (53)
suggested interaction between microbially synthesized humic acids and
polyvalent cations as an effective mechanism for the formation of
activated sludge. The fact that calcium ion positively effects floccu-
lation of cells of Zoogloea sp. which are capable of natural flocculation
may be indirect evidence of the presence of certain negatively charged
groups in the exopolymer necessary for chelation.
The discovery of smooth and rough colony-forming bacteria among strains
of Zoogloea came too late to permit a detailed commentary on these
organisms. It can be said that the presence of the two cell types in
test cultures quite likely contributed heavily to the variation observed
in flocculation experiments; a predominance of smooth type cells result-
ing in poor flocculation and, conversely, rough cells enhancing the
flocculence of cultures. We have noted during several years of handling
Zoogloea strains that freshly isolated cultures produced very little
turbidity in the growth medium. However, with continuous subculturing,
cultures became progressively more turbid and, in view of current
observations on rough-smooth cell types, it may be that the loop trans-
fer of culture encouraged the selection of smooth cells, since, generally,
subculture inocula contained dispersed cells rather than floes. In
this regard, we recommend that freshly isolated cultures, presumed to
be Zoogloea sp., be preserved by lyophillization and working cultures
be frequently plated and examined for the appearance of rough and smooth
type colonies whereupon each form may be maintained as desired. We
have only begun to screen our collection of Zoogloea strains for rough
and smooth colony forming cells. At this time, three cultures have been
found to contain each cell type. Although rough forms of the bacteria
very distinctly form cohesive floes, it is not known if these are the
predominant type in nature or to what degree they may revert to smooth
type cells. Although smooth cells are, essentially, nonflocculating,
they are capable of aggregating to some extent in quiescenct conditions
and it may be that they simply do not produce sufficient exopolymer
precursor and enough active enzyme to rapidly synthesize insoluble
polymer to produce much floe. Such deficiencies in smooth cells may be
the reason why calcium ion is not effective in their flocculation, that
is, sufficient negatively charged groups are not present at the surface
of the polymer to bind calcium ion and form tightly bridged cell
aggregates.
It is difficult to speculate on the importance of the rough and smooth
cells in the development of activated sludge since it is not known for
certain if Zoogloea spp. have an active role in the formation of natural
sludge floes. On the other hand, assuming that zoogloea-forming
bacteria are dominant among the polymer-producing sludge microflora and
that smooth and rough types freely exist there, it is possible that the
transition and behavior of the cells may influence the floe structure
76
-------
and, consequently, the physical stability and settleability of the
sludge solids. In addition, an abundance of polymer-producing cells in
the sludge may contribute to extensive formation of sludge exopolymer,
thus, increasing the nonviable matter in the sludge solids. Consequently,
biochemical activity would diminish per unit of mixed liquor volatile
solids. Although the sludge exopolymer consists of biodegradable sugar
monomers, it cannot be construed that the exopolymer is readily decom-
posed by microorganisms since the difficult requirement to hydrolyze the
glycosidic bonds contributes to the refractivity of the insoluble
polymer. Nonzoogloeal, bacterial floes may be disintegrated to
individual cells by severe agitation whereas cells embedded in the
gelatinous matrix of a zoogloea are held together and may be totally
released only upon dissolving the exopolymer. Activated sludge contains
both nonzoogloeal and zoogloeal bacteria and very likely, these are not
uniformily distributed through out floes.
Deflocculation of activated sludge is not well understood although
nutrient deficiency may be a factor in its occurrence (56). Whatever
the mode of action, microorganisms are responsible for the properties
of activated sludge and it is the opinion of Busch and Stumm (9) that
natural bioflocculation produces bacterial aggregates which are much
less sensitive to shearing forces than floes artificially established
with the aid of synthetic polyelectrolytes.
It is not certain that reducing agents are able to enhance zoogloea
formation by Zoogloea strains in axenic culture through lowering of
the oxidation-reduction potential in culture fluids. Zoogloeal scums
appear to form in static cultures of mixed liquors as a consequence of
volatile acid production which is enhanced by reducing environments.
Several of the short chain fatty acids which are normally formed from
wastewater solids under anaerobic conditions have been found to be
suitable nutrients for Zoogloea sp. (66). Under natural conditions,
fatty acids may be generated in the form of depot products of anaerobic
microorganisms acting upon organic matter. Diffusion of the volatile
acids to regions of slight oxygen tension (microaerophilic zone) would
permit them to serve as substrates for bacteria existing there. Unz
and Dondero (65) have commented on the microaerophilic tendency of
Zoogloea sp. which may also grow under definitely aerobic conditions.
We are encouraged that the exopolymer which we isolated from domestic
activated sludge consisted of two amino sugars as did the exopolymer
of Zoogloea strains. Our Zoogloea strains do not produce a micro-
fibrilar or cellulase sensitive exopolymer as described in other studies
(17,24,25). We are of the opinion that several kinds of bacteria exist
in nature which are capable of producing zoogloeae and, in all prob-
ability, the chemical composition of the exopolymers vary considerably.
However, in discussing Zoogloea sp., we take the position that the
misidentification of bacteria presumed to be Zoogloea sp. has occurred
77
-------
regularly in the past and will continue in this vein until the taxonomic
status of the genus Zoogloea and its members is clearly defined. A
reexamination of the genus Zoogloea and its species has been called for
by Zvirbulis and Hatt (71). Contrary to suggestions by Friedman et al.
(25), fibrilar polymers are not produced by all floe-forming bacteria
and the entrappment of bacteria in fibrils cannot be regarded as the
general basis upon which to explain natural bacterial flocculation.
Furthermore, the property of exocellular microfibril synthesis is not
universal among Zoogloea species and does not appear to be a useful
taxonomic character of these organisms. The Zoogloea strains which we
used for the major part of this study had the capacity to form finger-
like zoogloeae in axenic culture and had been thoroughly characterized
(65,66,68). These bacteria best represent the organisms responsible
for forming typical, finger-like zoogloeae in wastewaters and the data
obtained in this study should be treated accordingly.
78
-------
Section VIII
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61. Trevelyan, W. E., Procter, D. P., and Harrison, J. S., "Detection
of Sugars on Paper Chromatograms," Nature, 166, pp 444-445 (1950).
62. Ueda, S. and Earle, R. L., "Microflora of Activated Sludge,"
Journal General and Applied Microbiology, 18, pp 239-248 (1972).
63. Unz, R. F., "The Isolation and Characterization of the
Predommint Bacteria in Naturally Occurring Zoogloea ramigera
Colonies," Ph.D. Thesis, Rutgers University, New Brunswick,
New Jersey (1965).
83
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64. Unz, R. F., "Neotype Strain of Zoogloea ramigera Itzigsohn. Request
for an Opinion," International Journal Systematic Bacteriology, 21^
pp 91-99 (1971).
65. Unz, R. F. and Dondero, N. C., "The Predominant Bacteria in Natural
Zoogloeal Colonies. I. Isolation and Identification," Canadian
Journal of Microbiology, _13, pp 1671-1682 (1967).
66. Unz, R. F. and Dondero, N. C., "The Predominant Bacteria in Natural
Zoogloeal Colonies. II. Physiology and Nutrition," Canadian
Journal of Microbiology, 13, pp 1683-1694 (1967).
67. Unz, R. F. and Dondero, N. C., "Nonzoogloeal Bacteria in Wastewater
Zoogloeas," Water Research, 4_, pp 575-579 (1970).
68. Unz, R. F. and Farrah, S. R., "Use of Aromatic Compounds for Growth
and Isolation of Zoogloea," Applied Microbiology, 23, pp 524-530
(1972).
69. van Gils, H. W., Bacteriology ^f Activated Sludge, IG-TNO Report
No. 32. The Hague: Research Institute for Public Health Engineer-
ing (1964).
70. Wallen, L. L. and- Davis, E. N., "Biopolymers of Activated Sludge,"
Environmental Science & Technology. 6_t pp 161-164 (1972).
71. Zvirbulis, E. and Hatt, H. D., "Status of the Generic Name
Zoogloea and its Species," International Journal Systematic
Bacteriology, 17, pp 11-21 (1967) .
84
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Section IX
LIST OF PUBLICATIONS
1. Unz, R. F. and Farrah, S. R. , "Use of Aromatic Compounds for Growth
and Isolation of Zoogloea," Applied Microbiology, 23, pp 524-530
(1972).
85
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Section X
GLOSSARY
Activated sludge- A consortium of microorganisms, principally bacteria,
assembled in flocculent form and developed through aeration of wastewaters,
Antibody- A specific proteinaceous substance synthesized in the body of
an animal in response to the presence of an antigen.
Antigen- A foreign matter which when introduced into the body of an
animal elicits the production of antibody.
Antiserum- Blood serum which contains antibodies.
Cinematography- Motion picture photography.
Exopolymer- An extracellular, macromolecular substance of microbial
origin which constitutes the chemical basis of the gelatinous matrix of
a zoogloea.
Finger-like zoogloea- A peculiar, elongated zoogloea in which the
resident bacteria are generally arranged parallel to the longitudinal
axis of the zoogloea.
Floe- A flaky or granular aggregation of microorganisms which may be
cohesive or diffuse and may or may not be zoogloeal
Fluorescent antibody- Antibody which has been conjugated with a
fluorescent dye to assist in the location of antigen-antibody complexes
with the use of ultraviolet illumination and the microscope.
Mixed liquor- The mixture of activated sludge and wastewater which
is aerated in the activated sludge wastewater treatment process.
Mixed liquor suspended solids- The suspended solids, mostly activated
sludge, present in mixed liquor
Oxidation-reduction (redox) potential- A measure of the intensity level
of a system to donate (reduction) or accept (oxidation) electrons.
Serology- A field of study specializing in serum and serum reactions.
Zoogloea- A structure which consists of microorganisms embedded in a
confining gelatinous matrix. Not a generic epithet.
Zoogloea ramigera- Epithets for the genus and species of a legitimate
bacterium.
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TECHNICAL REPORT DATA ,
(Please read Instructions on the reverse before completing)
. REPORT NO.
EPA 670/2-74-018
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
BACTERIAL ZOOGLOEA FORMATION
5. REPORT DATE
April 1974
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Richard F. Unz and Samuel R. Farrah
9. PERFORMING ORG \NIZATION NAME AND ADDRESS
Department of Civil Engineering
Pennsylvania State University
University Park, Pennsylvania 16802
10. PROGRAM ELEMENT NO.
1BB043/21ASR/003
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
National Environmental Research Center
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Activated sludge floes suspended in wet mounts on microscope slides were observed
to sprout, finger-like, bacterial zoogloeae as a consequence of the outgrowth of
bacteria from floes. The rate of extension of finger-like zoogloeae was typically
5.1 to 15.0 |j,m per hr and mean cell doubling time was estimated to be approximately
2 hrs. Photomicrographic and fluorescent antibody studies revealed that the bacterial
zoogloeae consisted of the progeny of specific zoogloea-forming bacteria. Purified
exopolymers of Zoogloea strains and domestic activated sludge contained two amino
sugars, one of which was identified as glucosamine. Zoogloea exopolymer was not
fibrilar or cellulosic and contained approximately 17 to 19 per cent amino sugar and
about one per cent hexoses, uronic acids and ether soluble substances on a dry weight
of polymer basis. Amino sugar production was found to parallel zoogloea formation by
Zoogloea sp. Calcium ion appeared to augment flocculation of bacterial cells capable
of undergoing natural coalescence. Two cell types, described as rough and smooth
colony-forming, were found in some strains of Zoogloea. Rough cells readily floccu-
lated in agitated cultures whereas smooth cells produced relatively turbid cultures
under similar growth conditions. A predominance of one of the two types could
influence the degree of flocculation by Zoogloea cultures.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
*Activated sludge
*Bacteria
Flocculating
Microphotography
Polymers
Scum
Zoogloea sp.
Zoogloea ramigera
Zoogloea
Zoogloeal bacteria
Flourescent antibody
13B
06F
8. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
99
-87-
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
irU.S.Government Printing Office: 1974 — 757-582/5315 Region 5-i
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